Novel proteins within the type E botulinum neurotoxin complex

ABSTRACT

The invention features a polypeptide complex synthesized by bacteria of the genus Clostridia that contains the serotype E  botulinum  neurotoxin and five neurotoxin associated polypeptides having molecular weights of about 118, 80, 65, 40, and 18 kDa. respectively. The complex is useful in the treatment of diseases or conditions that are caused by excessive release of acetylcholine from presynaptic nerve terminals.

[0001] This application claims priority from U.S. application Ser. No.08/889,354, filed Jul. 8, 1997, now pending, which claims priority fromU.S. Provisional Application Serial No. 60/021,348, filed Jul. 8, 1996now abandoned.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This work was supported by grants from the United StatesDepartment of Agriculture (94-37201-1167) and the United StatesDepartment of Defense (NRL-ERDC-N00014-92-K-2007). The U.S. governmentmay have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to novel proteins that form acomplex with the type E botulin neurotoxin.

[0004] Various strains of the bacterium Clostridium, including C.botulinum, C. baratii, and C. butyricum, synthesize different serotypesof the potent neurotoxin botulin, which causes a form of food poisoningknown as botulism. C. botulinum synthesizes seven different serotypes ofbotulin, which are designated by the letters A through G. Humans andother animals come into contact with these neurotoxins most frequentlyby consuming food that is improperly stored in a way that permits growthof anaerobic bacteria. Foods that are typically tainted with botulininclude low acid canned meats and vegetables, preserved meats and fish,and pasteurized processed cheese spreads (Fogeding, In FoodborneMicroorganisms and Toxins: Developing Methodology, M. D. Pierson and N.Sterns, Eds., Marcel Dekker, Inc., New York, N.Y., 1986; Kautter et al.,J Food Prot. 42:784-786, 1979).

[0005] Another form of botulism, infant botulism, is thought to becaused by consumption of ubiquitous spores of C. botulinum along withfood (Simpson, In Botulinum Neurotoxin and Tetanus Toxin, AcademicPress, San Diego, Calif., 1989). These spores may colonize the infantintestine, germinate, and produce the neurotoxin. Similarly, spores thatgain access to deeply wounded tissue may germinate and produceneurotoxin within the wound.

[0006] Once present in the body, botulin neurotoxins cause muscleparalysis by blocking the release of acetylcholine from cholinergicnerve endings (DasGupta et al., Biochemistry and Pharmacology ofBotulinum and Tetanus Neurotoxins, In Perspective in Toxicology, A. W.Bernheimer, Ed., Wiley, New York, N.Y., 1977). Death may be caused byparalysis of the respiratory muscles.

[0007] The nucleotide sequences of the genes encoding all of thedifferent serotypes of the neurotoxin are known (Binz et al., J. Biol.Chem. 265:9153-9158, 1990; Campbell et al., J. Clin. Microbiol.31:2255-2262, 1993; East et al., FEMS Microbiol. Lett. 96:225-230. 1992;Hauser et al., Nucl. Acids Res. 18:4924, 1990; Whelan et al., Eur. J.Biochem., 204:657-667, 1992; and Whelan et al., Appl. Environ.Microbiol. 58:2345-2354, 1992). These genes are coordinately regulatedwith those encoding proteins that form complexes with the variousserotypes of botulin (Fujii et al., J. Gen. Microbiol. 139:79-83, 1993;and Nukina et al. In Botulinum and Tetanus Neurotoxins, B. R. DasGupta,Ed., Plenum Press, New York, N.Y., 1993). The A and B type neurotoxinsare associated with at least five other proteins, called “neurotoxinbinding proteins” or NAPS, while the type E neurotoxin has been found inassociation with only one other protein (Sugii et al., Infect. Immunol.12:1262-1270, 1975; Sakaguchi, Pharmac. Ther. 19:165-194, 1983; Schantzet al., Microbiol. Rev. 56:80-99, 1992; and Singh et al., J ProteinChem. 14:7-18, 1995).

[0008] The proteins that associate with the type A neurotoxin play acritical role in the food poisoning process by protecting the neurotoxinfrom the acids and proteolytic enzymes present in the gastrointestinaltract. For example, it is known that the oral toxicity of the intacttype A neurotoxin complex is 43,000 times greater than the oral toxicityof isolated and purified type A neurotoxin (Sakaguchi, Pharmac. Ther.19:165-194, 1983). The proteins associated with other serotypessimilarly “protect” the neurotoxin, but to a lesser degree.

SUMMARY OF THE INVENTION

[0009] The invention is based on the discovery that the type E botulinumtoxin exists in a complex that includes the toxin and five otherpolypeptides termed neurotoxin associated proteins (NAPs). Thisdiscovery is contrary to the prior assertions of those in the field, whobelieved that the toxin was associated with only one other polypeptide,a neurotoxin binding protein (NBP) having a molecular weight ofapproximately 118 kDa.

[0010] Accordingly, the invention features a substantially purepolypeptide complex that includes a Clostridium botulinum neurotoxin andmore than one Clostridium botulinum type E neurotoxin associatedpolypeptide. The polypeptides of the invention include the newlydiscovered NAPs, which have molecular weights of approximately 80, 65,40, and 18 kDa, and substantially pure antibodies that specifically bindto one or more of these polypeptides, or complexes of one or more of theNAPs with type E botulinum toxin, or toxins from other Clostridiumbotulinum serotypes including A, B, C, D, F, and G.

[0011] The following peptide sequences have been obtained: (1)MKQAFVFEFD (SEQ ID NO:1), from the 18 kDa polypeptide; (2) MRINTNINSM(SEQ ID NO:2), from the 40 kDa polypeptide; (3) MQTTTLNWDT (SEQ IDNO:3), from the 65 kDa polypeptide; and (4) TNLKPYIIYD (SEQ ID NO:4),from the 80 kDa polypeptide. In addition, the complete amino acidsequence of the 18 kDa polypeptide has been obtained and is shown inFIG. 8 (SEQ ID NO:5).

[0012] The compositions of the invention (e.g., the novel NAPs andantibodies that specifically bind to them) can be used to detect theserotype E neurotoxin complex in a sample. For example, one can contactthe sample with an antibody that specifically binds a NAP, or with a NAPthat binds the neurotoxin itself (as described in the Examples, below,the NAP having an approximate molecular weight of 80 kDa binds directlyto the type E neurotoxin) and detect, if present, antibody-bound type Eassociated polypeptide or NAP-bound type E neurotoxin. The presence ofthese antibody complexes indicates the presence of serotype E neurotoxinin the sample. The detection methods of the invention can be used toexamine virtually any type of sample, including samples of foodstuffs,or biological samples, such as gastrointestinal, blood, or tissuesamples obtained from a vertebrate animal.

[0013] The novel compositions of the invention also provide the basisfor methods of treating patients who suffer from a disease or conditionsassociated with excessive release of acetylcholine from presynapticnerve terminals. The patient is treated by administration of atherapeutically effective amount of a polypeptide complex that containsthe serotype E neurotoxin (or other serotype toxin) and more than oneNAP, e.g., one or more of the 80, 65, 40, and 18 kDa NAPs, and/or the118 kDa polypeptide. The conditions associated with excessiveacetylcholine release include undesirable contractions of smooth orskeletal muscle cells, which can, in turn, cause spasmodic torticollis,essential tremor, spasmodic dysphonia, charley horse, strabismus,blepharospasm, oromandibular dystonia, spasms of the sphincters of thecardiovascular, gastrointestinal, or urinary systems, or tardivedyskinesia. Excessive release of acetylcholine can also cause profusesweating, lacrimation, or mucous secretion. Patients who could benefitfrom the methods described herein include those who suffer fromspasticity that occurs secondary to another event such as brainischemia, traumatic injury of the brain or spinal cord, tensionheadache, or pain (e.g., pain caused by sporting injuries or arthritis).

[0014] In addition, the novel compositions of the invention can beformulated as a vaccine and administered to an animal in an amountsufficient to confer a degree of protection against serotype E (orother) neurotoxin.

[0015] Administration of a purified neurotoxin complex (e.g., of thetype E (or other) neurotoxin and more than one of the new NAPs), asdescribed below, is superior to administration of the neurotoxin alonebecause, within the complex, the neurotoxin is more stable and thus,longer-lasting. This feature minimizes the frequency of administrationand thereby reduces any risk, discomfort, or inconvenience that thepatient may experience.

[0016] The type E complex is a superior therapeutic agent, relative tothe other botulinum serotypes, because the activity of the type Eneurotoxin can be enhanced 100-fold by treatment with trypsin, whichbreaks the bonds between the two polypeptide chains that constitute theneurotoxin. Therefore, application of the type E neurotoxin complex canbe controlled by trypsinization, in a way that allows graded release ofthe neurotoxin from the complex. This unique mechanism provides morecontrolled and longer-lasting effects than would otherwise be possible.

[0017] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0018] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is an elution profile obtained by applying an extract ofE-type producing C. botulinum to a DEAE-Sephadex A-50 ion-exchangecolumn (A278 is absorbance at 278 nm).

[0020]FIG. 2 is a photograph of a polyacrylamide gel. Lane 1 was loadedwith the material that eluted in the first peak of FIG. 1. Lane 2 wasloaded with molecular weight standards. Lane 3 was loaded with materialeluted from a G-200 column (see FIG. 5).

[0021]FIG. 3 is an elution profile obtained by applying the type Ebotulinum neurotoxin complex to a Sephadex G-100 column.

[0022]FIG. 4 is an elution profile obtained by applying the type Ebotulinum neurotoxin complex eluted from a Sephadex G-100 column to aSephadex G-200 column.

[0023]FIG. 5 is an elution profile of the complex formed between type Ebotulinum neurotoxin and the 80 kDa component of the associated proteincomplex.

[0024]FIG. 6 is a photograph of an SDS-polyacrylamide gel. The materialin the first and second peaks of the elution profile shown in FIG. 5 waselectrophoresed in lanes 1 and 2, respectively.

[0025]FIG. 7 is a three-dimensional plot generated by light scatteringanalysis of the type E botulinum complex. The first and second series ofpeaks were generated with 105 nm and 225 nm diameter particles,respectively.

[0026]FIG. 8 is a representation of the amino acid sequence of a type Ebotulin NAP having a molecular weight of approximately 18 kDa (SEQ IDNO:5).

[0027]FIG. 9 is a schematic diagram of the genomic organization of C.botulinum neurotoxin serotypes C, A and B, F, and E.

DETAILED DESCRIPTION

[0028] Contrary to the general understanding in the field, the type Ebotulinum toxin complex consists in vivo of the neurotoxin, which has amolecular weight of about 150 kDa, and five (not one, as previouslybelieved) polypeptides that form a complex with the neurotoxin. Thesefive polypeptides have molecular weights of approximately 1118, 80, 65,40, and 18 kDa. Those of skill in the art will recognize that themeasured or apparent molecular weight of a polypeptide can varydepending, for example, on the number of glycosylated residues, and evenon the method used to determine the molecular weight. Accordingly, themeasured molecular weights of the NAPs of the invention can vary. Forexample, the measured molecular weight of the approximately 80 kDapolypeptide can vary between 70 and 90 kDa; the measured molecularweight of the approximately 65 kDa polypeptide can vary between 60 and70 kDa; the measured molecular weight of the approximately 40 kDapolypeptide can vary between 35 and 45 kDa; and the measured molecularweight of the approximately 18 kDa polypeptide can vary between 15 and21 kDa.

[0029] The novel polypeptides discovered in the type E botulinumneurotoxin complex can be used in a number of ways. First, they can beused to detect the presence of the type E botulin toxin in a sample,such as a food sample or a sample of biological tissue, or to generateantibodies that can be used in analogous detection methods. For example,if a patient is exposed to the neurotoxin, the NAPs and NAP-bindingantibodies of the invention provide the means (through direct bindingdetection systems or antibody-based detection systems) for rapid andreliable diagnosis. The NAPs, in their naturally occurring complex withthe type E neurotoxin (or complexed individually or in groups with typeE or other neurotoxins), are also useful in treating diseases associatedwith excessive release of acetylcholine from cholinergic nerve terminalsand, in addition, they can be used to generate vaccines forimmunization. These uses are described in further detail below.

[0030] Polypeptides of the Invention

[0031] The polypeptides of the invention include NAPs having molecularweights of approximately 80, 65, 40, and 18 kDa (alone or in anycombination, particularly in a combination that includes the type E (orother) botulin neurotoxin, and/or the neurotoxin binding protein (NBP)having a molecular weight of approximately 118 kDa), and antibodies thatspecifically bind to one or more of these NAPs or NAP complexes.

[0032] The invention encompasses full-length NAPs as well as fragmentsthat correspond to functional domains of the NAPs (e.g., fragments thatbind to polypeptides in the type E botulin complex and help to increasethe stability of the neurotoxin in vitro or in vivo, or fragments thatare antigenic (i.e., that elicit the production of antibodies)). TheNAPs of the invention, and fragments thereof, can have the sequence of awild-type NAP, or can contain a mutation (including deletions,additions, or substitutions of one or more amino acid residues).Preferably, the mutant polypeptides retain at least 50%, 75%, or atleast 95% or more of the biological activity of the correspondingwild-type polypeptide. It is a straightforward matter to compare thebiological activities of the mutant and wild-type polypeptides. Theycan, for example, be used in side-by-side tests for lethality inrodents. If an equivalent number of animals are killed regardless ofwhether they receive a particular dose of a type E complex containingwild-type or mutant NAPs, the mutant NAPs would be said to retain thebiological activity of the wild-type NAPs. If only half as many animalsdie following administration of a complex containing a mutant NAP, thenthe mutant would be said to retain half the biological activity of thewild-type NAP. Those of skill in the art will be aware of numerous waysin which biological activities can be fairly compared.

[0033] Mutant NAPs that contain a substitution of one or more amino acidresidues can be made purposefully or randomly (e.g., by using routinetechniques of recombinant DNA engineering or random mutagenesis,respectively). Amino acid substitutions may be purposefully changed toaffect the polarity, charge, solubility, hydrophobicity, hydrophilicity,or amphipathic nature of the residues involved. Not only may the mutantpolypeptides retain biological activity, this activity could beincreased. For example, a mutant NAP could be made to bind with a higheraffinity to the type E botulin neurotoxin.

[0034] Polypeptides in which the NAPs are fused to an unrelated protein(e.g., a protein that can be easily detected or quantitated) are alsoconsidered within the scope of the invention.

[0035] The polypeptides of the invention can be purified from anaturally-occurring source, chemically synthesized (e.g., see Creighton,In Proteins: Structures and Molecular Principles, W.H. Freeman & Co.,New York, N.Y., 1983), or produced by recombinant DNA technology usingtechniques well known in the art for expressing nucleic acids. Thesemethods can, for example, be used to construct expression vectorscontaining a NAP-encoding sequence, and appropriate transcriptional andtranslational control signals.

[0036] “Substantially pure” polypeptides are polypeptides inpreparations in which they represent at least 60% by weight of thepreparation. When one or more NAPs are present in a complex, forexample, in a complex with the type E botulin neurotoxin, asubstantially pure preparation will consist of at least 60%, by weight,the polypeptides of the given complex. Preferably, preparationscontaining one or more NAPs are at least 75%, at least 90%, or even atleast 99%, by weight, the polypeptide(s) of interest. Purity can bereadily measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0037] The amino acid sequence of the NAP having a molecular weight ofapproximately 18 kDa has been determined (FIG. 8; SEQ ID NO:5), andpartial sequences are described herein for the NAPs having molecularweights of approximately 80 (SEQ ID NO:4), 65 (SEQ ID NO:3), and 40 kDa(SEQ ID NO:2). It is well within the abilities of those of ordinaryskill in the art to obtain additional sequence information from thepartial sequences disclosed herein. For example, PCR technology can beused to isolate full length NAP cDNA sequences as follows. First, RNAcan be isolated, following standard procedures, from an appropriatecellular or tissue source (e.g., a bacterium of the genus Clostridia),and reverse transcribed using an oligonucleotide primer specific for themost 5′ end of the amplified fragment. This oligonucleotide primes firststrand synthesis. The resulting RNA/DNA hybrid can then be “tailed” withguanines using a standard terminal transferase reaction, and the hybridcan be digested with RNAse H. Second strand synthesis can then be primedwith a poly-C primer. The sequence of the amplified fragment can then beobtained using any number of routine procedures.

[0038] Production of Antibodies Against the Type E Neurotoxin AssociatedProteins

[0039] Antibodies that specifically bind to one or more epitopes of aNAP, or fragments or derivatives thereof, are also considered within thescope of the present invention. An antibody is said to “specificallybind” to a polypeptide when it recognizes and binds to that polypeptide,but not to other molecules in a sample (e.g., a biological sample thatincludes type E neurotoxin associated polypeptides). The antibodies ofthe invention can be polyclonal, monoclonal, humanized, chimeric, orsingle chain antibodies, or Fab fragments, F(ab′)2 fragments, fragmentsproduced by a Fab expression library, anti-idiotype (anti-Id)antibodies, and epitope-binding fragments of any of the types ofantibodies listed above.

[0040] A variety of standard methods can be used to generate antibodiesagainst the type E neurotoxin associated proteins. For example, the typeE neurotoxin associated proteins, either individually or in theircomplexed forms, can be administered to an animal, such as a rat, mouse,or rabbit, to induce the production of polyclonal antibodies.Alternatively, antigenic fragments of the individual polypeptides can beused to generate polyclonal antibodies. Various adjuvants can be used toincrease the immunological response to an antigen, depending on the hostspecies. These adjuvants include Freund's (complete or incomplete)adjuvant, mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Potentiallyuseful human adjuvants are known to include BCG (bacile Calmette-Guerin)and Corynebacterium parvum.

[0041] In addition, antibodies according to the invention can bemonoclonal antibodies e., antibodies from a homogenous population thatrecognize a particular antigen) that are generated by using eitherindividual serotype E NAPs, the intact type E complex, or complexes ofthe neurotoxin with the NBP and any one or more of the new NAPs. Suchmonoclonal antibodies can be prepared using standard hybridomatechnology (see; e.g., Kohler et al. Nature 256:495, 1975; Kohler etal., Eur. J. Immunol. 6:292, and 6:511, 1976; Hammerling et al., InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981;Kruisbeck et al., Hornbeck et al., and Yokoyama, In Current Protocols inImmunology, Vol. 1, New York, John Wiley & Sons, Inc., 1994). Monoclonalantibodies can be of any immunoglobulin class, including the IgG, IgM,IgE, IgA, and IgD classes, and any subclass thereof. The hybridomaproducing the mAb of the invention can be cultivated in vitro or invivo.

[0042] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-7955,1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature314:452-454, 1985) by splicing the genes from a mouse antibody moleculeof appropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region.

[0043] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-426,1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; andWard et al., Nature 334:544-546, 1989) can be adapted to produce singlechain antibodies against NAPs. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

[0044] Antibody fragments that recognize specific epitopes can also begenerated by known techniques. For example, such fragments include, butare not limited to: the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275-1281, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0045] The binding specificity and activity of purified anti-type E (orother serotypes) complex antibodies, such as those described above, canbe confirmed by testing their ability to interfere with the biologicalactivity of the neurotoxin and/or the complex. This ability can betested by adding the antibodies to any number of standard in vitroassays in which the release of acetylcholine from presynaptic nerveterminals can be monitored. These assays include preparations ofdifferent neuromuscular junctions, such as the mouse phrenicnerve-hemidiaphragm, the mouse plantar nerve-lumbrical muscle, and chickciliary ganglion-iris muscle preparations (Bandyopadhyay et al., J.Biol. Chem. 262:2660-2663, 1987); Bittner et al., J. Biol. Chem.264:10354-10360, 1989; Clark et al., J. Neurosci. Methods 19:285-295,1987; and Lomneth et al., Neurosci. Lett., 113:211-216, 1990). Thebinding specificity and activity of any given antibody is tested bydetermining whether that antibody effectively blocks the action of typeE neurotoxin complex applied at the neuromuscular junction.

[0046] Polypeptide-Based Detection Systems for Type E NeurotoxinAssociated Proteins

[0047] The NAPs described herein have a variety of uses, including thedetection of type E neurotoxin. The type A neurotoxin remains associatedwith its protein complex both in bacterial culture medium and in naturalcases of food poisoning (Sakaguchi, Pharmac. Ther. 19:165-194, 1983).Given this evidence, it is likely that the 118 kDa binding protein andthe other four, lower molecular weight members of the type E complexalso remain associated with the cognate toxin in vitro and in vivo. Inaddition, neurotoxin associated proteins have been shown to be moreimmunogenic than the neurotoxin itself (Singh et al., Toxicon34:267-275, 1996).

[0048] Antibodies generated against any one of the NAPs or combinationsthereof can also be used to detect the type E neurotoxin complex usingvarious standard methods. For example, the antibodies can be used with afiber optic-based biosensor, as described herein, which uses anevanescent wave from a tapered optical fiber for signal discrimination.This antibody-based “sandwich” immunoassay detection system can detectbotulinum toxin much more quickly than any method currently available,but other immunoassay methods can be used. The actual signal collectionperiod with the biosensor is less than one minute. Detection isaccomplished using a two-step sandwich immunoassay. Antibody-boundoptical fibers are incubated in a solution of type E complex, and asignal is generated when the fiber-bound complex binds a fluorescentlylabeled antibody (see, Ogert et al., Anal. Biochem. 205:306-312, 1992;and Singh et al., In Natural Toxins II, B. R. Singh and A. Tu, Eds.,Plenum Press, pp. 498-508, 1996).

[0049] One of the problems historically associated with sandwichimmunoassays is that the first antibody (here, the antibody bound to theoptical fiber) and the second antibody (here, the antibody added todetect the fiber-bound complex), compete for the same epitope on theneurotoxin. To circumvent this problem, two antibodies can be used. Thefirst specifically binds to one portion of the neurotoxin or one NAP ofthe type E complex, which will be attached to the fiber, and a secondspecifically binds to either a second portion of the neurotoxin or asecond member of the polypeptide complex, which would specificallyrecognize the fiber-bound complex.

[0050] Any polypeptide (be it a NAP or an antibody of the invention) canbe detectably labeled to facilitate the detection of the type E botulincomplex. For example, the polypeptides can be conjugated with aradio-opaque or other appropriate compound, such as a fluorescentcompound, that can be brought into contact with a sample that maycontain the botulin complex. Alternatively, the polypeptide can belinked to an enzyme and used in an enzyme immunoassay (EIA; Voller etal. J. Clin. Pathol. 31:507-520, 1978; Butler, Methods Enzymol.73:482-523, 1981). The enzyme that is bound to the antibody will reactwith an appropriate substrate, e.g., a chromogenic substrate, in such amanner as to produce a chemical moiety that can be detected, forexample, by spectrophotometric, fluorimetric, or visual means. Enzymesthat can be used to detectably label a polypeptide include malatedehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeastalcohol dehydrogenase, beta-galactosidase, alkaline phosphatase,ribonuclease, and urease.

[0051] Preparation and Administration of A Neurotoxin Vaccine

[0052] The invention also includes a vaccine composition containing atype E (or other serotype) neurotoxin and more than one type Eneurotoxin associated polypeptide (or immunogenic fragment or derivativethereof) and a pharmaceutically acceptable diluent or carrier, such asphosphate buffered saline or a bicarbonate solution (e.g., 0.24 MNaHCO₃). The carriers and diluents used in the invention are selected onthe basis of the mode and route of administration, and standardpharmaceutical practice. Suitable pharmaceutical carriers and diluents,as well as pharmaceutical necessities for their use, are described inRemington's Pharmaceutical Sciences. An adjuvant, for example, a choleratoxin, Escherichia coli heat-labile enterotoxin (LT), or a fragment orderivative thereof having adjuvant activity, may also be included in thevaccine composition of the invention.

[0053] Skilled artisans can obtain further guidance in the preparationof a vaccine for type E neurotoxin complex in Singh et al. (Toxicon27:403-410, 1990). Briefly, approximately 1.5 mg of the type E complexis added to approximately 10 ml of 0.05 M sodium citrate buffer (pH 5.5)and dialyzed against 0.39% formaldehyde at 3.0° C. for 7 days. Theformaldehyde-containing buffer is replaced every day with fresh buffersolution. The detoxified neurotoxin (toxoid or vaccine) is dialyzedagainst 0.1 M sodium phosphate buffer (pH 7.4) without formaldehyde fortwo days with several changes of buffer.

[0054] The amount of vaccine administered will depend, for example onthe particular vaccine antigen, whether an adjuvant is co-administeredwith the antigen, the type of adjuvant co-administered, the mode andfrequency of administration, and the desired effect (e.g., protection ortreatment), as can be determined by one skilled in the art. In general,the vaccine antigens of the invention are administered in amountsranging between, for example, 1 μg and 100 mg. If adjuvants areadministered with the vaccines, amounts of the polypeptide vaccineranging between, for example, 1 ng and 1 mg can be used. Administrationis repeated as necessary, as can be determined by one skilled in theart. For example, a priming dose can be followed by three booster dosesat weekly intervals.

[0055] Treatment with Polypeptides in the Type E Neurotoxin Complex

[0056] Any disease or discomfort associated with an exaggerated releaseof acetylcholine from a presynaptic nerve terminal can be treated withthe purified or isolated type E botulinum neurotoxin complex describedherein, or with complexes formed of other serotype toxins combined withone or more of the NAPS described herein. Those of skill in the art areaware of the normal parameters for acetylcholine release and of thenormal range of physiological function that is produced when anappropriate amount of this neurotransmitter is released onto a musclefrom adjacent nerve terminals. An exaggerated release of acetylcholinewould be any level of release that exceeds the normal parameters andcauses aberrant physiological function. The diseases or conditionsassociated with an exaggerated release of acetylcholine can involvespasms of either smooth or skeletal muscle cells. More specifically,these diseases or conditions include spasmodic torticollis, essentialtremor, spasmodic dysphonia, charley horse, strabismus, blepharospasm,oromandibular dystonia, spasms of the sphincters of the cardiovascular,gastrointestinal, or urinary systems, and tardive dyskinesia, which mayresult from treatment with an anti-psychotic drug such as THORAZINE® orHALDOL®.

[0057] For example, an adult male patient suffering from tardivedyskinesia resulting from treatment with an antipsychotic drug can betreated with 50-200 units (defined below) of Botulinum type E complex bydirect injection into the facial muscles. Within three days, thesymptoms of tardive dyskinesia, i.e., orofacial dyskinesia, athetosis,dystonia, chorea, tics and facial grimacing are markedly reduced.

[0058] Spasticity that occurs secondary to brain ischemia, or traumaticinjury of the brain or spinal cord, are similarly amenable to treatment.

[0059] In instances where the postsynaptic target is a gland, nerveplexus, or ganglion, rather than a muscle, the type E complex can beadministered to control profuse sweating, lacrimation, and mucoussecretion. For example, an adult male patient with excessive unilateralsweating can be treated by administering 0.01 to 50 units of type Ebotulinum complex to the gland nerve plexus, ganglion, spinal cord, orcentral nervous system. Preferably, the nerve plexus or ganglion thatmalfunctions to produce the excessive sweating is treated directly.Administration of type E neurotoxin complex to the spinal cord or brain,while feasible, may cause general weakness.

[0060] Other conditions that can be treated include tension headache andpain caused by sporting injuries or arthritic contractions. Ifnecessary, overactive muscles can be identified with electromyography(EMG).

[0061] While it is expected that the methods of the invention will mostcommonly be applied to human patients, domestic pets (such as dogs andcats) and livestock (such as cows, sheep, and pigs) can also be treatedwith the compositions described herein.

[0062] Administration of Polypeptides within the Type E NeurotoxinComplex

[0063] The dose of type E neurotoxin complex administered to a patientwill depend generally upon the severity of the condition, the age,weight, sex, and general health of the patient, and the potency of thetoxin, which is expressed as a multiple of the LD₅₀ value for the mouse.

[0064] The dosages used in human therapeutic applications are roughlyproportional to the mass of muscle to be treated. Typically, the doseadministered to the patient is from about 0.01 to about 1,000 units, forexample, about 500 units. A unit is defined as the amount of type Eneurotoxin (or type E neurotoxin complex) that kills 50% of a group ofmice (typically a group of 18-20 female mice that weigh on average 20grams). The dosage is adjusted, either in quantity or frequency, toachieve sufficient reduction in acetylcholine release to afford relieffrom the symptoms of the disease or condition being treated.

[0065] Physicians, pharmacologists, and other skilled artisans are ableto determine the most therapeutically effective treatment regimen, whichwill vary from patient to patient. The potency of botulinum toxin andits duration of action means that doses are administered on aninfrequent basis. Skilled artisans are also aware that the treatmentregimen must be commensurate with questions of safety and the effectsproduced by the toxin.

[0066] Typically, the type E neurotoxin complex is suspended in aphysiologically acceptable solution, such as normal saline, and isadministered by an intramuscular injection. Prior to injection, carefulconsideration is given to the anatomy of the muscle group, in an attemptto inject the toxin complex into the area with the highest concentrationof neuromuscular junctions. If the muscle mass is not very great, theinjection can be performed with extremely fine, hollow, teflon-coatedneedles and guided by electromyography. The position of the needle inthe muscle should be confirmed prior to injection of the toxin, andgeneral anesthesia, local anesthesia, or other sedation may be used atthe discretion of the attending physician, according to the age andparticular needs of a given patient and the number of sites to beinjected.

[0067] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES

[0068] Production of Botulinum Toxin by Cell Culture

[0069] Generally, to obtain botulinum toxin in large amounts, complexedmedia containing combinations of meat hydrolysate, casein hydrolysate,yeast autolysate, yeast extract, and glucose supplemented with one ormore reducing agents are used (Sakaguchi. Pharmac. Ther. 19:165-194,1983). Vegetables autoclaved in saline also provide an excellent culturemedium, supporting toxin production by type A- and type B-producingbacteria to a similar extent as laboratory media. Glucose must be addedfor type E- and type F-producing bacteria to grow in boiled vegetables(Sugii et al., J. Food Safety 1:53-65, 1977). The optimum temperaturefor toxin production by C. botulinum is generally regarded as 20-3° C.

[0070] Type E C. botulinum Produces a Complex Including Five NeurotoxinAssociated Proteins For this series of experiments, C. botulinum type E(available from the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., 20852 (U.S.A.); Type E Clostridium botulinumAccession Nos. 9564, 17786, 17852, 17854, and 17855) was grown for 4days in 15 ml cooked meat medium. Stock cultures were prepared accordingto standard methods and stored at −20° C.

[0071] The stock culture was activated at 30° C. for approximately 25hours and then transferred to a growth medium containing 2.0%Trypticase-peptone, 1.0% glucose, 0.025% sodium thioglycolate (BBLMicrobiology Systems, Cockeysville, Md.), and 0.5% yeast extract (Difco)adjusted to pH 6.5. When large culture volumes (8 liters) were used a12% glucose solution was autoclaved and added to the broth, which wasseparately prepared and then autoclaved for 1 hour. The culture wasincubated for 60-65 hours, and cells were collected by centrifugation.An extract from the cells was prepared at 20° C. by stirring with 0.2 Mphosphate buffer (pH 6.0). The resulting suspension was saturated with(NH₄)₂SO₄; 39 g/ml) and stored at 4° C.

[0072] DEAE-Sephadex Chromatography

[0073] The crude extract described above was precipitated andredissolved in 35 ml of 0.05 M sodium phosphate buffer (pH 5.5). Theresulting solution was clarified by centrifugation and chromatographedon a DEAE-Sephadex A-50 ion-exchange column (Pharmacia). The sample waseluted from the column with 0.05 M sodium citrate at pH 5.5. It isimportant that the pH of the buffer is maintained at 5.5. The firstprotein peak (FIG. 1) was pooled as type E complex.

[0074] In contrast to previous reports (Sugii et al., Infect. Immunol.12:1262-1270, 1975; Sakaguchi, Pharmac. Ther. 19:165-194, 1983; Schantzet al., Microbiol. Rev. 56:80-99. 1992; Singh et al., J Protein Chem.14:7-18, 1995), a total of five different proteins were found in thecomplex in addition to the 150 kDa type E botulinum neurotoxin.Specifically, the material constituting the first peak of the elutionprofile described above (and shown in FIG. 1) was analyzed bySDS-polyacrylamide gel electrophoresis. Six proteins, having molecularweights of approximately 150 (the neurotoxin), 118, 80, 65, 40, and 18kDa, were apparent (FIG. 2).

[0075] Size Exclusion Chromatography

[0076] To further confirm the nature of the type E complex, the proteinswere analyzed on size exclusion chromatographic columns. The type Ecomplex that eluted from the DEAE Sephadex A-50 column was concentratedto 5 mg/ml and applied to a Sephadex G-100 column (1.8×92 cm or 2.6×82cm, 0.05 M sodium citrate buffer, pH 5.5). The resulting elution profilerevealed one peak in the void volume (FIG. 3). Three of the proteinspresent are clearly less than the exclusion limit of the column andthus, should elute separately from the void volume. Since this did notoccur, and all six proteins continued to elute in one peak, it wasconcluded that the proteins are bound together in a complex. A similarresult was obtained following chromatography on a Sephadex G-200 column(FIG. 4), further confirming that the six proteins form a singlecomplex.

[0077] One of the neurotoxin associated proteins in the type E complex,the 80 kDa protein, was purified and studied for its ability to re-forma complex with pure type E neurotoxin. After combining the 150 kDaneurotoxin and the 80 kDa associated protein, the elution profileobtained from a Sephadex G-200 column revealed a major peak containingboth the 80 kDa protein and the type E neurotoxin, and aminor peakcontaining excess uncomplexed 80 kDa protein (FIG. 5). The materialeluted in each of the two peaks was electrophoresed on anSDS-polyacrylamide gel (FIG. 6), which confirmed the content of theprotein(s) in each peak.

[0078] The 80 kDa Neurotoxin Associated Protein Specifically Binds TypeE Neurotoxin

[0079] A kinetic binding study performed with an optical fiber-basedbiosensor revealed that the type E neurotoxin could bind directly to the80 kDa type E neurotoxin associated protein, rather than associateindirectly with the neurotoxin via another polypeptide in the complex.The 80 kDa polypeptide was tested for its ability to bind directly tothe neurotoxin at pH 7.5 and at pH 5.7. The type E botulinum neurotoxinwas first immobilized, and the purified 80 kDa NAP was labeled withTRITC (Tetramethyl-rhodamine-isothiocyanate; Molecular Probes, Eugene,Oreg.) as described in Ogert et al. (Anal. Biochem. 205:306-312, 1992),except that unreacted TRITC was removed by dialysis, rather than gelfiltration. The binding experiments were carried out by blocking theexposed sites on the optical fiber with 2% BSA (at room temperature) andincubating them with TRITC-labeled 80 kDa polypeptide (5 mg/ml) that hadbeen equilibrated with phosphate buffered saline (PBS; at pH 5.7 or pH7.5). The initial rate of binding was calculated based on the signalincrease within the first 60 seconds.

[0080] Subsequent polypeptide binding rates at pH 7.5 and 5.7 were 4.01and 8.42 μV/minute, respectively, suggesting that the interactionbetween the neurotoxin and the 80 kDa polypeptide is significant at pH7.5, and considerably higher at pH 5.7. Therefore, the 80 kDapolypeptide could play a role in protecting the type E neurotoxin fromthe acidic conditions present in the gastrointestinal tract.

[0081] These results are consistent with the known behavior of thebotulinum neurotoxin complex, which dissociates at alkaline pH levels.Thus, the associated binding polypeptides can be used as a specificbinding partner to “capture,” and thereby detect, the neurotoxin. Thismethod would effectively detect the neurotoxin wherever it exists, to atleast some degree, free from the complex or, at least, free from the 80kDa neurotoxin binding protein. The 80 kDa NAP also complexes with TypeA neurotoxin.

[0082] Sequence Analysis of Proteins in the Type E Neurotoxin Complex

[0083] Partial amino acid sequences of the novel polypeptides in theserotype E neurotoxin complex were obtained as follows. Approximately 10picomoles of the purified type E neurotoxin complex were dissolved in abuffer consisting of 0.5 M sucrose, 15% SDS (sodium dodecyl sulfate),312.5 mM Tris, and 10 mM EDTA, and electrophoresed on a 12.5%SDS-acrylamide gel using a Mini-PROTEAN II™ electrophoresis cell(Bio-Rad Laboratories, Hercules, Calif.). The electrophoresis wasperformed in running buffer (2 g/L Tris base, 14.4 g/L glycine, 1 g/LSDS and 0.1 mM sodium thioglycolate, pH 8.3) under a constant voltage(200 V). The protein was then electrotransferred from the gel to a PVDFmembrane in a Twobin buffer (25 mM Tris, 192 mM glycine and 20%methanol) using a Mini Trans-Blot electrophoretic transfer cell™(Bio-Rad Laboratories, hercules, Calif.). The transfer was carried outovernight at 60 volts in an ice bath. To visualize the protein bands,the membrane was stained with 0.025% Coomassie Blue R250 in 40% methanoland destained with 50% methanol. The proteins bound to the PVDFmembranes were sequenced at Baylor College of Medicine (Houston, Tex.)using Applied Biosystem Model 473A protein sequencer™ (Foster City,Calif.).

[0084] The following peptide sequences were obtained: (1) MKQAFVFEFD(SEQ ID NO:1), from the 18 kDa protein; (2) MRINTNINSM (SEQ ID NO:2),from the 40 kDa protein; (3) MQTTTLNWDT (SEQ ID NO:3), from the 65 kDaprotein; and (4) TNLKPYIIYD (SEQ ID NO:4), from the 80 kDa protein.These sequences were compared with those of known proteins associatedwith neurotoxin types A, B, and C. This analysis failed to reveal anyregions of homology with the type A associated proteins of C. botulinum.

[0085] Genomic Organization

[0086] The genomic organization of NAPs of type E C. botulinum wasinvestigated using oligonucleotide primers having sequencescomplementary to the N-terminal amino acid sequences. Using combinationsof sense and antisense primers (the sequences of which were derived fromthe amino acid sequences shown in SEQ ID NOs:1-4 using standardtechniques) in a conventional polymerase chain reaction (wherechromosomal DNA from bacteria of the genus Clostridia served as thetemplate), the topography of the 18 and 80 kDa NAPs were revealed. Bothof these genes are transcribed in the opposite direction to that of theneurotoxin and NBP (i.e., the 118 kDa neurotoxin binding protein) genes(FIG. 9). In addition, there is an open reading frame between the NBPand the 18 kDa NAP that has the same transcriptional direction as theNBP. This protein sequence did not match with any of the proteins thathave been identified as NAPs of type E C. botulinum. In addition torevealing the orientation of various NAP-encoding genes, theseexperiments revealed that the genes are clustered together within thegenome.

[0087] Analysis of Type E Neurotoxin Complex by Light Scattering

[0088] To characterize the type E neurotoxin complex as a whole, lightscattering experiments were performed on material purified byDEAE-Sephadex A-50 chromatography (1.5 mg/ml). Analysis was performed ona Malvern 4700 PCS Autosizer System (Malvern Instruments Inc.) equippedwith an eight-bit, 136 channel correlator capable of variable timeexpansion. The laser light source was model INNOVA 70-5 argon laser(Coherent, Calif.). A 514.5 nm line was employed in single operationmode with 1.0 watt power output.

[0089] Initial results from light scattering experiments suggested thatthe complex exists in two forms, as 600 and 2000 kDa molecular weightspecies (FIG. 7). The combined molecular weight of the proteins in thetype E neurotoxin complex observed on polyacrylamide gels is 468 kDa.The difference between these two predicted sizes could be due either tovariation in the folding of the complex or to the existence ofoligomeric forms of some of the proteins in the complex.

[0090] NAPs Protect the Toxic Activity of Botulin Neurotoxin from Heat

[0091] To investigate the role of NAPs in protecting the type E botulincomplex from heat, the neurotoxin alone and the intact complex were eachheated to 60° C. for 15 minutes, then cooled, and incubated with“cracked” PC 12 cells that had been grown in tritiated norepinephrineand stimulated to release norepinephrine by calcium. The term “cracked”is used to describe cells that have been treated with a mechanicaldevice so that their integrity is somewhat disrupted; this technique isdescribed in Lomneth et al., J. Neurochem. 57:1413-1421 (1991). Thepercentage of norepinephrine released into the culture medium was thenassessed, because the toxin blocks release of norepinephrine from thecracked cells.

[0092] In control experiments, the neurotoxin alone and the intactcomplex were each incubated with cracked PC12 cells, but not subjectedto the 60° C. heat treatment. The control botulin neurotoxin complex(i.e., a complex that had not been heat-treated) reduced the percentageof norepinephrine release from a normal 56.1±0.8% in buffer-treated (nocomplex, no toxin) cells, to 22.5+0.3% in cells treated by theneurotoxin complex at a final concentration of 50 μg/ml.

[0093] In spite of heat treatment (and potential denaturation), theheat-treated complex, at the same concentration of 50 μg/ml, was stillable to block, i.e., reduce, the percentage of norepinephrine release to34.1±0.9%. The type E neurotoxin, when not heated and used alone (i.e.,without NAPs) blocked (or reduced) the percentage of norepinephrinerelease to 21.0±0.6% at 50 μg/ml, whereas heat-treated neurotoxin wasnot able to substantially block the norepinephrine release (the percentof norepinephrine released was 51.9±1.9%). These data clearly suggestthat the presence of NAPs is effective in providing functional stabilityto the type E botulinum complex.

[0094] To determine whether the NAPs provided protection to the botulincomplex by preventing heat-induced unfolding of polypeptides in thecomplex, or by assisting in proper refolding of heated toxin, theheat-induced unfolding of purified neurotoxin and the neurotoxin complexwere analyzed by monitoring their circular dichroism (CD) signal at 222nm. This method has been used successfully to monitor unfolding ofproteins (see, e.g., Fahnestock et al., Science 258:1658-1662, 1992; andLehrer and Qian, J. Biol. Chem. 265:1134-1138, 1990). The midpointunfolding temperature (Tm) for the neurotoxin was 54° C. whereas the Tmfor the large (new) type E complex was 70° C. The Tm for a neurotoxincomplex consisting of the 118 kDa NBP and the type E botulin neurotoxinis between these two values (Sakaguchi, Pharmac. Ther. 19:165-194, 1983;Singh et al., J Protein Chem. 14:7-18, 1995). These observations clearlyindicate that the loss of type E botulin neurotoxin activity afterheating at 60° C. is due to the unfolding of the toxin, whereas no suchunfolding occurs in the presence of NAPs.

[0095] An observation made while conducting this set of experiments wasthat the type E botulinum complex is equally (or more) effective inblocking the neurotransmitter release from PC 12 cells compared to thepure neurotoxin (without NAPs), although the total effectiveconcentration of the neurotoxin in the complex was only about a third ofthe pure toxin concentration (1111 nM vs. 333 nM). This suggests thatthe NAPs actually activate the neurotoxin, which would be consistentwith our hypothesis that the folding of the type E complex can bealtered by NAPs.

Other Embodiments

[0096] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 5 10 amino acids amino acid linear peptide 1 Met Lys Gln Ala Phe ValPhe Glu Phe Asp 1 5 10 10 amino acids amino acid linear peptide 2 MetArg Ile Asn Thr Asn Ile Asn Ser Met 1 5 10 10 amino acids amino acidlinear peptide 3 Met Gln Thr Thr Thr Leu Asn Trp Asp Thr 1 5 10 10 aminoacids amino acid linear peptide 4 Thr Asn Leu Lys Pro Tyr Ile Ile TyrAsp 1 5 10 144 amino acids amino acid linear peptide 5 Met Glu Leu LysGln Ala Phe Val Phe Glu Phe Asp Glu Asn Leu Ser 1 5 10 15 Ser Ser SerGly Ser Ile His Leu Glu Lys Val Lys Gln Asn Cys Ser 20 25 30 Pro Asn TyrAsp Tyr Phe Lys Ile Thr Phe Ile Asp Gly Tyr Leu Tyr 35 40 45 Ile Lys AsnLys Ser Gly Val Ile Leu Asp Lys Tyr Asp Leu Lys Asn 50 55 60 Val Ile SerLeu Val Ala Leu Lys Arg Asp Tyr Leu Ser Leu Ser Leu 65 70 75 80 Ser AsnAsn Lys Gln Ile Lys Lys Phe Lys Asn Ile Lys Asn Lys His 85 90 95 Leu LysAsn Lys Phe Asn Leu Tyr Val Ile Asn Glu Asp Ile Glu Lys 100 105 110 ArgIle Thr Lys Asn Gly Ile Leu Glu Glu Val Ile Leu Asn Lys Met 115 120 125Leu Leu Ser Ile Leu Leu Gly Asn Glu Glu Asn Leu Leu Gln Ile Ser 130 135140

What is claimed is:
 1. A substantially pure polypeptide complexcomprising a Clostridium botulinum neurotoxin and more than oneClostridium botulinum type E neurotoxin associated polypeptide.
 2. Acomplex of claim 1, wherein the neurotoxin associated polypeptide has amolecular weight of approximately 80 kDa and comprises the amino acidsequence TNLKPYIIYD (SEQ ID NO:4).
 3. A complex of claim 1, wherein theneurotoxin associated polypeptide has a molecular weight ofapproximately 65 kDa and comprises the amino acid sequence MQTTTLNWDT(SEQ ID NO:3).
 4. A complex of claim 1, wherein the neurotoxinassociated polypeptide has a molecular weight of approximately 40 kDaand comprises the amino acid sequence MRINTNINSM (SEQ ID NO:2).
 5. Acomplex of claim 1, wherein the neurotoxin associated polypeptide has amolecular weight of approximately 18 kDa and comprises the amino acidsequence MKQAFVFEFD (SEQ ID NO:1).
 6. A complex of claim 1, wherein theneurotoxin associated polypeptide has a molecular weight ofapproximately 18 kDa and comprises the amino acid sequence shown in FIG.8 (SEQ ID NO:5).
 7. A substantially pure Clostridium botulinum serotypeE neurotoxin associated polypeptide.
 8. The polypeptide of claim 7,wherein the neurotoxin associated polypeptide has a molecular weight ofabout 80 kDa.
 9. The polypeptide of claim 8, wherein the neurotoxinassociated polypeptide comprises the amino acid sequence TNLKPYIIYD (SEQID NO:4).
 10. The polypeptide of claim 7, wherein the neurotoxinassociated polypeptide has a molecular weight of about 65 kDa.
 11. Thepolypeptide of claim 10, wherein the neurotoxin associated polypeptidecomprises the amino acid sequence MQTTTLNWDT (SEQ ID NO:3).
 12. Thepolypeptide of claim 7, wherein the neurotoxin associated polypeptidehas a molecular weight of about 40 kDa.
 13. The polypeptide of claim 12,wherein the neurotoxin associated polypeptide comprises the amino acidsequence MRINTNINSM (SEQ ID NO:2).
 14. The polypeptide of claim 7,wherein the neurotoxin associated polypeptide has a molecular weight ofabout 18 kDa.
 15. The polypeptide of claim 14, wherein the neurotoxinassociated polypeptide comprises the amino acid sequence MKQAFVFEFD (SEQID NO:1).
 16. The polypeptide of claim 14, wherein the neurotoxinassociated polypeptide comprises the amino acid sequence shown in FIG. 8(SEQ ID NO:5).
 17. A substantially pure antibody that specifically bindsto a Clostridium botulinum type E neurotoxin associated polypeptidehaving a molecular weight of approximately 80, 60, 45, or 18 kDa, or toa complex of any two or more of said neurotoxin associated polypeptides.18. A substantially pure antibody that specifically binds to apolypeptide complex of claim
 1. 19. A method of detecting a serotype Eneurotoxin complex in a sample, the method comprising: (a) contactingthe sample with an antibody of claim 17, and (b) detectingantibody-bound polypeptide, if any, in the sample, the presence ofantibody-bound polypeptide indicating the presence of serotype Eneurotoxin in the sample.
 20. The method of claim 19, wherein the sampieis a foodstuff.
 21. The method of claim 19, wherein the sample is agastrointestinal, blood, or tissue sample obtained from a vertebrateanimal.
 22. A method of treating a patient who is suffering from adisease or condition associated with excessive release of acetylcholinefrom presynaptic nerve terminals, the method comprising administering tothe patient a therapeutically effective amount of a polypeptide complexof claim
 1. 23. The method of claim 22, wherein the excessiveacetylcholine release causes undesirable contraction of smooth orskeletal muscle cells.
 24. The method of claim 22, wherein the excessiverelease of acetylcholine causes profuse sweating, lacrimation, or mucoussecretion.
 25. A method of treating a patient who is suffering fromspasticity occurring secondary to brain ischemia, or traumatic injury ofthe brain or spinal cord, the method comprising administering to thepatient a therapeutically effective amount of a polypeptide complex ofclaim
 1. 26. A method of treating a patient who is suffering fromtension headache or pain, the method comprising administering to thepatient a therapeutically effective amount of a polypeptide complex ofclaim
 1. 27. A vaccine comprising a polypeptide complex of claim
 1. 28.A method of vaccinating an animal against serotype E neurotoxin, themethod comprising administering to the animal an effective amount of thevaccine of claim
 27. 29. A vaccine comprising a polypeptide of claim 7.30. A method of detecting a Clostridium botulinum serotype E neurotoxinin a sample, the method comprising: (a) contacting the sample with aClostridium botulinum type E neurotoxin associated polypeptide (NAP) ofclaim 7 that specifically binds a serotype E botulinum neurotoxin andthereby forms a NAP-neurotoxin complex, and (b) detecting theNAP-neurotoxin complex, if any, in the sample, the presence of a complexindicating the presence of serotype E neurotoxin in the sample.
 31. Acomplex of claim 1, comprising the neurotoxin and neurotoxin associatedpolypeptides having molecular weights of about 80 kDa, 65 kDa, 40 kDa,and 18 kDa.
 32. The complex of claim 1, comprising the neurotoxin andneurotoxin associated polypeptides having molecular weights of about 118kDa, 80 kDa, 65 kDa, 40 kDa, and 18 kDa.